Cerebrospinal fluid proteomics identifies calcyphosine and follistatin-like 1 as exploratory candidate proteins of interest in hydrocephalus

Background

Hydrocephalus comprises etiologically heterogeneous disorders that converge on ventricular enlargement but may be associated with distinct protein-abundance patterns within the cerebrospinal fluid (CSF) compartment. This exploratory study compared CSF proteomic profiles in post-hemorrhagic hydrocephalus (PHH) and idiopathic normal pressure hydrocephalus (iNPH) to characterize CSF protein-abundance patterns associated with these two hydrocephalus and identify proteins for further validation.

Methods

Cerebrospinal fluid samples from 11 participants, including five patients with PHH, three with iNPH, and three non-hydrocephalus controls, were analyzed using Olink proximity extension assay proteomics. Normalized protein expression values were assessed by quality-control analysis, differential expression analysis, and functional annotation using Gene Ontology, KEGG, Reactome, InterPro, Disease Ontology, and STRING-based protein interaction analyses. Differentially expressed proteins were screened using nominal p values, with false discovery rate adjustment calculated for statistical interpretation. Calcyphosine (CAPS) and follistatin-like 1 (FSTL1) were further assessed by ELISA in an expanded cohort.

Results

All samples passed quality-control criteria. Compared with the non-hydrocephalus control group, both PHH and iNPH showed predominantly downregulated CSF proteomic profiles, with different exploratory protein-abundance patterns. PHH showed relative CAPS elevation together with reduced proteins related to neuronal structural maintenance, synaptic signaling, axon guidance, immune communication, and extracellular regulation. Functional annotation analyses identified overrepresented terms related to inflammatory signaling, cytokine-receptor interaction, cell adhesion, calcium-related signaling, lysosomal clearance, glycan remodeling, and neural pathways. In iNPH, FSTL1 was relatively increased, whereas proteins involved in synaptic function, axon guidance, cell adhesion, growth-factor signaling, extracellular matrix organization, and cellular stress responses were decreased. Enrichment analyses highlighted neural connectivity, receptor-associated signaling, inflammatory pathways, proteostasis, glycosaminoglycan metabolism, and cilium- or centrosome-related processes. ELISA reproduced the direction of the proteomic findings, showing higher CSF CAPS levels in PHH and higher CSF FSTL1 levels in iNPH than in the non-hydrocephalus control group.

Discussion

The PHH and iNPH share a ventricular phenotype but exhibit distinct CSF proteomic signatures. CAPS and FSTL1 may represent exploratory proteins of interest within different hydrocephalus-related annotation contexts. These findings require validation in larger, independent, longitudinal cohorts before clinical biomarker inferences are made.